2667 Introduction It is well established that the embryonic environment can influence the growth of the embryo in many species. In particular, the effect of temperature on subsequent growth has been studied for a long time in fish (reviewed by Johnston, 2006) but also, more recently, in the development of avians (Maltby et al., 2004) and reptiles (Booth, 1998; Booth, 2006). The effects of incubation temperature have been examined on the growth of long bones in chick (Brookes and May, 1972) and on muscle in fish and turkeys (Johnston, 2006; Maltby et al., 2004). Whilst it is clear that temperature can influence the development both of muscle and of bone, the mechanisms by which this is occurring are unclear. In addition to the general developmental biology interest in studying the effects of environment on the development of the chick musculoskeletal system, there are important welfare implications in this work. A huge and growing number of chicks are bred each year for their meat, with 860·million chicks per year in the UK (Defra, 2006). The majority are intensively farmed in order to elicit the highest weight gain in the shortest time. There have been a number of reports showing the correlation between rapid growth rates and the incidence of leg disorders (Kestin et al., 2001; Kestin et al., 1992), with further reports showing that this affects the behaviour of the birds and their ability to walk (Vestergaard and Sanotra, 1999; Weeks et al., 2000). It would therefore be of great potential benefit to manipulate the growth and development of support tissues such as leg muscle and bone by making simple changes to incubation regimes such that chickens are better able to support greater weights and thereby reduce the incidence of abnormalities. A problem with investigating the effects of temperature in chicks is that the scope for altering the incubation temperature of poultry eggs is relatively small. In turkeys, increasing the incubation temperature by 1°C throughout the duration of incubation causes a significant reduction in hatchability, whereas shorter periods of around 4·days spent at the higher temperature do not cause decreases in viability (French, 2000). In chick embryos, the period between embryonic day 4 (ED4), roughly HH23 on the Hamburger and Hamilton staging series (Hamburger and Hamilton, 1951), and ED7 (roughly HH30) corresponds to the time when the primary muscle fibres have been laid down in the trunk and is just prior to the formation of the secondary muscle fibres, which begins at ED8 (Crow and Stockdale, 1986; Lee et al., 2004). It is a time when there is a great deal of proliferation among myoblasts, followed by differentiation. It is also the time that the limb buds form and grow and when the undifferentiated cells of the limb mesenchyme start to differentiate as muscle, cartilage and skin (reviewed in Christ and Brand-Saberi, 2002). From ED5, the limb muscle masses undergo cleavage, and the final muscle pattern is apparent by ED7.5 or HH30 (Kardon, 1998; Pautou et al., 1982). Thus, it is a critical time in limb development, and as such we hypothesize that intervention at this time would give the greatest changes to the development of limb tissues. The chick embryo, developing in the egg, is an ideal system in which to investigate the effects of incubation environment on the development of the embryo. We show that raising the temperature of the eggs by just one degree, from 37.5°C to 38.5°C, during embryonic days (ED) 4–7 causes profound changes in development. We demonstrate that embryonic movement is significantly increased in the chicks raised at 38.5°C both during the period in which they are at the higher temperature but also 4·days after their return to the control temperature. Concomitant with this increase in embryonic activity, the embryos raised at higher temperature grow to significantly heavier weights and exhibit significantly longer leg bones (tibia and tarsus) than the controls from ED12 onwards, although mineralization occurs normally. Additionally, the number of leg myonuclei is increased from ED12 in the embryos raised at the higher temperature. This is likely to promote greater leg muscle growth later in development, which may provide postural stability to the chicks posthatch. These changes are similar to those seen when drugs are injected to increase embryonic activity. We therefore believe that the increased embryonic activity provides a mechanism that can explain the increased growth of leg muscle and bone seen when the eggs are incubated for 3·days at higher temperature. Key words: chick, movement, temperature, growth, incubation, muscle, bone. Summary The Journal of Experimental Biology 210, 2667-2675 Published by The Company of Biologists 2007 doi:10.1242/jeb.005751 In ovo temperature manipulation influences embryonic motility and growth of limb tissues in the chick (Gallus gallus) Christina L. Hammond*, Biggy H. Simbi and Neil C. Stickland Department of Veterinary Basic Sciences, The Royal Veterinary College, Royal College Street, London, NW1 0TU, UK *Author for correspondence (e-mail: chhammond@rvc.ac.uk) Accepted 16 May 2007 THE฀JOURNAL฀OF฀EXPERIMENTAL฀BIOLOGY